Wacharapluesadee et al. Virology Journal (2015) 12:57 DOI 10.1186/s12985-015-0289-1

SHORT REPORT Open Access

Diversity of coronavirus in bats from EasternThailandSupaporn Wacharapluesadee1*, Prateep Duengkae2, Apaporn Rodpan1, Thongchai Kaewpom1,Patarapol Maneeorn3, Budsabong Kanchanasaka3, Sangchai Yingsakmongkon1,4, Nuntaporn Sittidetboripat1,Chaiyaporn Chareesaen3, Nathawat Khlangsap2, Apisit Pidthong3, Kumron Leadprathom5, Siriporn Ghai1,Jonathan H Epstein6, Peter Daszak6, Kevin J Olival6, Patrick J Blair7, Michael V Callahan1,8 andThiravat Hemachudha1

Abstract

Background: Bats are reservoirs for a diverse range of coronaviruses (CoVs), including those closely related tohuman pathogens such as Severe Acute Respiratory Syndrome (SARS) CoV and Middle East Respiratory SyndromeCoV. There are approximately 139 bat species reported to date in Thailand, of which two are endemic species. Dueto the zoonotic potential of CoVs, standardized surveillance efforts to characterize viral diversity in wildlife areimperative.

Findings: A total of 626 bats from 19 different bat species were individually sampled from 5 provinces in EasternThailand between 2008 and 2013 (84 fecal and 542 rectal swabs). Samples collected (either fresh feces or rectal swabs)were placed directly into RNA stabilization reagent, transported on ice within 24 hours and preserved at −80°C untilfurther analysis. CoV RNA was detected in 47 specimens (7.6%), from 13 different bat species, using broadly reactiveconsensus PCR primers targeting the RNA-Dependent RNA Polymerase gene designed to detect all CoVs. Thirty sevenalphacoronaviruses, nine lineage D betacoronaviruses, and one lineage B betacoronavirus (SARS-CoV related) wereidentified. Six new bat CoV reservoirs were identified in our study, namely Cynopterus sphinx, Taphozous melanopogon,Hipposideros lekaguli, Rhinolophus shameli, Scotophilus heathii and Megaderma lyra.

Conclusions: CoVs from the same genetic lineage were found in different bat species roosting in similar or differentlocations. These data suggest that bat CoV lineages are not strictly concordant with their hosts. Our phylogenetic dataindicates high diversity and a complex ecology of CoVs in bats sampled from specific areas in eastern regions ofThailand. Further characterization of additional CoV genes may be useful to better describe the CoV divergence.

Keywords: Coronavirus, Bats, Diversity, Eastern, Thailand

BackgroundFollowing the Severe Acute Respiratory Syndrome(SARS) pandemic in 2002–03, caused by the SARS cor-onavirus (SARS-CoV), intensive surveillance has de-tected a great diversity of CoVs throughout the animalkingdom, especially in bats. The initial discovery of CoVsin bats was made in China following the SARS outbreak[1-5]. The emergence of the Middle Eastern Respiratory

* Correspondence: spwa@hotmail.com1World Health Organization Collaborating Centre for Research and Trainingon Viral Zoonoses, King Chulalongkorn Memorial Hospital, Faculty ofMedicine, Chulalongkorn University, Bangkok, ThailandFull list of author information is available at the end of the article

© 2015 Wacharapluesadee et al.; licensee BioMCreative Commons Attribution License (http:/distribution, and reproduction in any mediumDomain Dedication waiver (http://creativecomarticle, unless otherwise stated.

Syndrome (MERS) in 2012 renewed interest in bat-originated CoVs. The molecular investigation in SaudiArabia revealed one Taphozous perforatus bat whosevirus showed 100% nucleotide identity to the MERSvirus found in the human index case [6]. Other subse-quent studies have found MERS-related CoV lineagesfrom a variety of bat species globally [7-11]. CoVs aredivided into four genera: Alphacoronavirus and Beta-coronavirus which largely infect mammals; and Gam-macoronavirus and Deltacoronavirus which primarilyinfect avian species [12]. CoVs in bats are generally ofthe Alpha- and Betacoronavirus genus, and have beenidentified in bats of various species from around the

ed Central. This is an Open Access article distributed under the terms of the/creativecommons.org/licenses/by/4.0), which permits unrestricted use,, provided the original work is properly credited. The Creative Commons Publicmons.org/publicdomain/zero/1.0/) applies to the data made available in this

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world. Thailand is home to 139 different bat species, ofwhich two are endemic species including Hipposiderospendleburyi and Murina balaensis (new species ofgenus Murina) [13,14], however CoV surveillance hasonly been conducted on 25 (18%) of these species [15].The first report of bat CoVs in Thailand examined atotal of 256 fecal specimens and discovered 28 positivesamples in H. larvatus and H. armiger [15]. Recently ina study in Ratchaburi province, Thailand, we discoveredlineage C betacoronavirus in dry bat guano fertilizer,however the bat species was not identified as specimenswere collected from a mixed species roost [11]. As a re-sult of the risk CoVs pose to human health, ecologicalstudies of CoVs in bats are warranted, particularly tounderstand the baseline viral diversity circulating inwildlife hosts. Here we describe a comprehensive studyof CoV diversity and prevalence among bats in EasternThailand to explore CoV infections in bat populations.

MethodsBats were captured with permission from the Depart-ment of National Parks, Wildlife and Plant Conserva-tion. The Institutional Animal Care and Use Committeeat the University of California, Davis (protocol number:16048) approved the capture and sample collection. Bats’species were identified in the field by experienced Thaimammalogist (PD) based on their external morphologicalcharacteristics as described by Lekagul & McNeely[16,17]. Fresh bat fecal pellets were individually stored in0.5 ml of RNAlater® RNA Stabilization Reagent (Qiagen,Germany) while each rectal swab was placed into 1 ml ofNucliSens® Lysis Buffer (bioMérieux, France), and thenstored at −80°C until further analysis. Samples were exam-ined using broadly reactive consensus hemi-nested Re-verse Transcription PCR (RT-PCR) with degenerate PCRprimers designed to detect all CoV lineages, targeting theRNA-dependent RNA polymerase (RdRp) gene [18]. Amp-lification product was visualized using 2% agarose gel elec-trophoresis. The RdRP PCR product was sequenceddirectly using an automated ABI PRISM 377 model se-quencer or cloned using the pGEM®-T Easy Vector Sys-tem before sequencing. Initially, to assess clonal sequencediversity, ten individual clones from each specimenwere sequenced. Sequences were edited using Bio-editprogram. Sixty-three CoV sequences obtained from 47specimens (more than one sequence was found from 4specimens) were deposited in GenBank with accessionnumbers [KJ020577 to KJ020636, KJ652018, KJ868721and KJ868722]. Phylogenetic trees were constructedbased on 353 bp RdRp gene sequence, corresponding tonucleotides 14,355 - 14,707 in Human CoV 229E gen-ome (GenBank accession no. AF304460) using the max-imum likelihood method.

ResultsA total of 626 bats representing 19 species (Table 1)were sampled (84 fecal and 542 rectal swab specimens)between 2008 and 2013 from 6 locations in 5 of 7 prov-inces in Eastern Thailand. CoV RNA was detected in 47(7.6%) specimens (17 fecal samples and 30 rectal swabs)from 13 different bat species. Detection rates for batCoVs were 1.6% to 45% per site in 5 of the samplingsites (Figure 1). Phylogenetic analysis of nucleotide se-quences of 353 bp RdRp gene showed that 37 sampleswere members of the Alphacoronavirus genus and 10belonged to the Betacoronavirus genus. The phylogen-etic reconstruction showed 9 different clades of batCoVs (Figure 2). There were 6 clades in alphacorona-virus (clades 1–6), 2 in lineage D betacoronavirus (clades7 and 8) and 1 in lineage B betacoronavirus (clade 9).The six alphacoronavirus clades were divergent, butwere related to CoVs previously identified in bats fromChina, Bulgaria and Kenya [19-21]. The percent nucleo-tide similarity within each clade was calculated and is in-cluded in Figure 2.In our study, host restriction of CoVs was demon-

strated in clade 4 (CoV512) and clade 6 (HKU2). Clade4 CoV from Scotophilus heathii was clustered with batCoV512 previously found in S. kuhlii from China [4].Clade 6 CoV (BRT55555) found in Rhinolophus shameli,was clustered with HKU2 (R. sinicus) described in China[22]. This is in accordance with previous studies whichhave demonstrated that individual CoVs are associatedwith a single species or genus including Carollia, Eptesi-cus, Miniopterus, and Rhinolophus bats [4,19,20,23].We found evidence for species of CoV in almost every

clade in this study being shared by different bat speciesfrom different families. For example clades 1–3 CoVswere found in 3 bat families, in the Miniopterus magna-ter, M. schreibersii, M. pusillus, H. lekaguli, H. armigerand T. melanopogon. Similarly, clade 5 CoVs were foundin 6 bat species (5 genera, 5 families) from 4 differentsites, clade 7 CoVs were found in 3 bat species capturedfrom the same location and clade 8 CoVs were found inCynopterus sphinx (fruit bat) and the insectivorous H.lekaguli (Figure 2). Further, many CoVs species werefound in a single bat species such as the H. lekaguli, R.shameli, T. melanopogon, and S. heathii (Figure 2). Forexample, 10H. lekaguli bats roosting in the same colonywere found to harbor 2 lineage D betacoronaviruses and8 alphacoronaviruses.Seven CoVs in clade 7 from S. kuhlii, S. heathii and C.

sphinx were clustered in an independent lineage. Theseviruses (from 5 bats) had 99.15-100% identity of 119amino acids and differed from HKU9 by 16.11-16.95%(Figure 2). Further analyses using longer gene fragmentsand other genes from greater number of bats are re-quired for confirmation of this novel group.

Table 1 Bat species tested for coronaviruses

Family Species No. of positive/total‡ (%)

Sampling site (year)† CoV clade(s) [cluster]/(no. positive)

Pteropodidae Cynopterus brachyotis 1/9 (11.1) AA(2011*); TR(2011) 5 [HKU10]/(1)

Cynopterus sphinx 4/14 (28.6) AA(2011); RD(2008); TR(2011); CB(2012*) 7 [New cluster]/(2), 8 [HKU9]/(2)

Eonycteris spelaea 0/11 (0) AA(2011); TR(2011); CB(2012)

Rousettusamplexicaudatus

0/3 (0) SK(2011)

Emballonuridae Taphozouslongimanus

0/12 (0) RD(2008/2012)

Taphozousmelanopogon

2/123(1.6) RD(2012*/2013); CK(2012); SK(2011*/2012) 2 [HKU7]/(1), 5 [HKU10]/(1)

Hipposideridae Hipposideros armiger 2/140(1.4) CK(2012); RD(2008/2012*/2013); SK(2012) 1 [CoV1A/B]/(1), 5 [HKU10]/(1)

Hipposideroscineraceus

0/3 CK(2012)

Hipposideros larvatus 1/29(3.4) CK(2012); RD(2008/2012/2013*) 9 [SARS]/(1)

Hipposideroslekaguli

10/159(6.3) CK(2008*/2012*) 1 [CoV1A/B]/(2), 5 [HKU10]/(6), 8 [HKU9]/(2)

Macroglossinae Macroglossussobrinus

0/2(0) AA(2011); TR(2011)

Megadermatidae Megaderma lyra 1/2 (50) RD(2012*/2013) 5 [HKU10]/(1)

Rhinolophidae Rhinolophusshameli

2/20(10) CK (2012*) 5 [HKU10]/(1), 6 [HKU2]/(1)

Vespertilionidae Miniopterus magnater 6/30(20) CK (2012*) 1 [CoV1A/B]/(5), 2 [HKU7]/(2**)

Miniopterus pusillus 1/1(100) CK(2012*) 3 [HKU8]/(1)

Miniopterusschreibersii

12/53(22.6) CK(2008*/2012) 1 [CoV1A/B]/(3), 2 [HKU7]/(1), 3 [HKU8]/(8)

Myotis horsfieldii 0/4(0) CK(2012); CB(2012)

Scotophilus kuhlii 2/3(66.7) CB(2012*) 7 [New cluster]/(2)

Scotophilus heathii 3/8(37.5) CB(2012*) 4 [CoV 512]/(2), 7 [New cluster]/(1)

Total 47/626(7.5)

‡Samples were 84 fecal and 542 rectal swabs; *A positive location (and year) is indicated by an asterisk; First report of CoV in species (indicated in bold).†AA = Ang Aed, Chataburi; CB = Chonburi; CK = Chakan, Srakaew; RD = Rad, Chachongsao; SK = Sarika, Chantaburi; TR = Trat.**Sample No. BRT55593 (Miniopterus magnater) contained 2 different CoV species belong to clade 1 and 2.

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One specimen from M. magnater (BFE55593) wasfound to be co-infected with 2 CoV species. Further, threeindividual bats from 2 species (samples no. B128 [M.schreibersii], B311 [M. schreibersii], and B55700 [C.sphinx]) were found to be co-infected with multiplestrains of the same CoV species. Initial sequencing showedmultiple nucleotide peaks upon direct sequencing chro-matogram. These PCR products were cloned and 10 indi-vidual clones were sequenced in order to assess clonalsequence diversity. Analysis of 353 bp sequences revealedthe presence of sequence variants within single samples(also known as quasispecies [24]). There were 7, 4, 6, and3 different sequences obtained from samples no. B128,B311, BFE55593, and B55700, respectively. All sequencesfrom individual samples were clustered into the sameCoV species (i.e. represented CoV quasispecies) except inBFE55593, where 2 sequences were clustered to clade 2and the other 4 sequences were clustered into clade 1.

In addition, this is the first report describing the pres-ence of CoV RNA in 6 bat species including C. sphinx,T. melanopogon, H. lekaguli, R. shameli, S. heathii andMegaderma lyra, where the latter is the newly reportedbat family (Megadermatidae) found to harbor CoV.

Discussion and conclusionData from this study demonstrates that CoV infection inbats sampled in Eastern Thailand is not uncommon andinfection is distributed among a range of species. TheCoVs found in bats from this small region of Thailandwere genetically related to bat CoVs found in severalcountries from different regions of the world such asChina, Philippines, Kenya, Spain and Bulgaria [18-21,25].MERS-like CoV (previously found in environmentallysampled bat feces from Ratchaburi province, WesternThailand [11]) was not found in this study, despite sam-pling the bat genus Taphozous, a likely MERS-CoV

Figure 1 Areas in Eastern Thailand where samples were collected with CoV-positive bat species additionally named. Chakarn cave (CK, Dark blue)in Srakaeo province; Rad cave (RD, Pink) in Chachoengsao province; Chonburi province (CB, Red); Ang Aed (AA, Pale blue) and Sarika (SK, Green)caves in Chanthaburi province; and Trat province (TR, Orange). At CK site, bats were captured 4 times: in May 2008, July 2008, January 2012 andMay 2012; RD site, 5 times: May 2008, July 2008, January 2012, May 2012 and January 2013; SK site, 2 times: December 2011 and May 2012; AAsite, 1 time: December 2011; TR site, 1 time: December 2011. Between January and December 2012 at CB site, bats were captured monthly at 2local swine farms. Generally, bats were caught in mist nets or harp traps as they emerged from their roosts. At two sites (CB and TR), bats weretrapped during the night as they foraged near open orchards. The number of CoV positive bats [bracket] in each clade is indicated for each site.** Sample No. BRT55593 (Miniopterus magnater) contained 2 different CoV species belong to clade 1 and 2.

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reservoir found in Saudi Arabia [6], in our study. Furtherstudies on individual bat species in Western Thailand toidentify bat reservoirs for MERS- or SARS-like CoVs maybe justified. Phylogenetic analysis revealed close corre-lations between CoV/B56054 from H. larvatus andSARS-like CoV belonging to lineage B betacoronavirusfrom Rhinolophus in China [1]. This finding was in ac-cordance with the previous bat CoV study in Thailand[15]. However three bat species (H. lekaguli, M. lyra,and M. schreibersii) and 3 bat families (Pteropodidae,Emballonuridae, and Rhinolophidae) previously re-ported negative for CoV in Thailand [15], were positivefor CoVs in our study (Table 1).At several of our sites, we observed many different bat

species from different families roosting in the same caveand subsequently were found to be harboring the samebat CoV species. For example, H. lekaguli harbored CoVsof the same genetic lineage as Miniopterus CoV in clades1, and R. shameli in clade 5. Co-roosting of theses batsin an enclosed cave environment may have facilitatedthe exchange of viruses. Three bat species (S. kuhlii, S.

heathii and C. sphinx) captured from the same location(unknown roost) carried similar viruses clustered inclade 7. The spatial overlap at feeding areas and tempor-ary night roosts for S. kuhlii, S. heathii (insectivorous)and C. sphinx (nectarivorous) may have facilitated theexchange of viruses, as they may not necessarily be co-roosting diurnally and do not share the same direct foodsource. This data supports previous studies in Spain,China and South America, where different bat speciessampled within the same location carried similar viruses[4,25,26]. However, more research is needed to under-stand interspecific bat behavior and transmission poten-tial for these species with seemingly different diet andforaging patterns.Further, the H. armiger and T. melanopogon, which

roosted at a different colony from the Miniopterusbats, also harbored CoV of the same genetic lineage asthe Miniopterus CoV in clades 1 and 2 respectively.Similarly, HKU10-related CoVs (clade 5) were found in5 divergent bat families including Megadermatidae,Pteropodidae, Emballonuridae, Hipposideridae, and

Figure 2 (See legend on next page.)

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(See figure on previous page.)Figure 2 Phylogenetic trees of the coronavirus (CoV) RNA-dependent RNA polymerase (RdRp) gene at the nucleotide level. Maximum-likelihood tree ofa 353 bp fragment of the RdRp gene from bat CoVs found in this study are colored according to their roost (Dark blue = Chakarn cave, CK; Pink = Radcave, RD; Red = Chonburi province, CB; Pale blue = Ang Aed, AA; Green = Sarika cave, SK) and previously found in bats and other animals (black). ABulbul deltacoronavirus HKU11-934 was used as outgroup. Alignments were constructed using Multiple Alignment Fast Fourier Transform, MAFFT.Bootstrap values were determined using 1000 replicates via MEGA 5. The tree was visualised using the FigTree program, version 1.4.0. Taxa are namedaccording to the following pattern: identification code/strain or isolate/typical host/country/collection year/accession number. Cyn_bra, Cynopterusbrachyotis; Cyn_sph, Cynopterus sphinx; Tap_mel, Taphozous melanopogon; Hip_arm, Hipposideros armiger; Hip_lar, Hipposideros larvatus; Hip_lek,Hipposideros lekaguli; Meg_lyr, Megaderma lyra; Rhi_sha, Rhinolophus shameli; Min_mag, Miniopterus magnater; Min_pus, Miniopterus pusillus;Min_sch, Miniopterus schreibersii; Sco_kuh, Scotophilus kuhlii; Sco_hea, Scotophilus heathii. There were 7, 4, 6, and 3 different sequences obtained fromsamples no. B128 (B128-1 to B128-7), B311 (B311-1 to B311-4), BFE55593 (BFE55593-1 to BFE55593-6, and B55700 (B55700-1 to B44700-3), respectively.Representative sequences where the same exact CoV species (>99% nucleotide similarity) was found in different individuals of the same bat species atthe same site show in italic. Clades 1–6 of alphacoronavirus were categorized based on the CoVs previously reported in China; bat-CoV1A/B, −HKU7, −HKU8, −CoV512, −HKU10 and -HKU2, respectively while clade 7–8 and 9 of betacoronavirus were categorized based on HKU9 andSARS CoV, respectively. The percent nucleotide similarity within each clade is shown in parentheses under the clade name.

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Rhinolophidae (Figure 2). These HKU10- positive bat spe-cies, H. lekaguli (CK site), H. armiger (RD), C. brachyotis(AA), T. melanopogon (SK), M. lyra (RD), and R. shameli(CK), were from 4 different sampling sites. Interestingly,these viruses were closely related to HKU10 CoVs foundin Rousettus leschenaulti and H. pomona in China, whereinterspecies transmission between bats of different subor-ders was also demonstrated [27]. Further studies and dee-per characterization of these bat CoVs infecting differentbat species may provide additional insight to their hostrange and the evolutionary history of their interspeciestransmission. These findings indicate a greater diversityand higher ecological complexity of bat CoVs in EasternThailand than previously appreciated.Two or more different CoV clades/lineages were also

found circulating in the same bat species from the samesite, for example C. sphinx, H. armiger, H. lekaguli, R.shameli, M. schreibersii, M. magnater, and S. heathii,and from different roosts for T. melanopogon (Table 1and Figure 2). This CoV diversity may be associated withbat migration or bats from different species sharing for-aging sites. Previous studies also found evidence ofcross-species transmission in bats, for example Artibeuslituratus from Mexico [23], and R. sinicus [28] and R.leschenaulti from China [2].Interestingly, co-infection of divergent CoV lineages

was found in one bat (M. magnater, BFE55593), whichwas infected with 2 different CoV species, clustered inclades 1 (CoV 1A/B) and 2 (HKU7) (Figure 2). This find-ing is similar to a previous report from China, where co-infection of bat CoV 1B and HKU8 were detected in M.pusillus using species-specific RT-PCR assays [29]. Co-infection with different CoVs in the same host may fa-cilitate recombination between these CoVs. Furtherstudies of co-infection and CoV recombination within agiven bat host could improve our understanding on theevolution of CoVs, including specific mutations or re-combination events (e.g. involving the Spike gene), thatcould facilitate spillover to novel species.

In conclusion, phylogenetic analysis of our study re-vealed a high genetic diversity of CoVs and presence ofcross species dissemination in bats from the Eastern re-gion of Thailand. Finding of new viral reservoirs and theputative novel betacoronavirus lineage in this study em-phasizes the need for additional CoV surveillance. Ourdata can serve as an additional dataset to the global sur-veillance of emerging CoVs, which may include poten-tially harmful pathogens to human health. In order tohave a complete understanding of the ecology and trans-mission of CoV, a comprehensive analysis of bats acrosstheir migratory routes in Africa, Southeast Asia andAustralia should be conducted.

AbbreviationsCoV: Coronavirus; MERS: Middle East Respiratory Syndrome; SARS: SevereAcute Respiratory Syndrome; RT-PCR: Reverse transcription-polymerase chainreaction.

Competing interestsThe authors declare that they have no competing interests.

Authors’ contributionsSW and PD participated in the design of the study and drafted themanuscript. PD, PM, BK, CC, NK, AP and KL conducted sampling of bat fecalsamples. TK, SY, and carried out the molecular genetic studies. AR and SGparticipated in the sequence alignment and drafted the manuscript. TH, KJOand PJB wrote the draft of the manuscript. MVC, JHE and PD carried outrevision of the manuscript. All authors read and approved the finalmanuscript.

Authors’ informationThe GenBank accession numbers for the coronavirus sequences reported inthis paper are: KJ020577 – KJ020636, KJ652018, KJ868721 and KJ868722.

AcknowledgmentsThis study was supported by a research grant from the Thailand ResearchFund (RDG5420089), the Ratchadaphiseksomphot Endowment Fund ofChulalongkorn University (RES560530148-HR), Health and Biomedical ScienceResearch Program by National Research Council of Thailand (NRCT) andHealth System Research Institute (HSRI), the Research Chair Grant, theNational Science and Technology Development Agency (NSTDA), Thailand,the Naval Health Research Center (BAA-10-93) under the CooperativeAgreement no. W911NF-11-2-0041, and the USAID Emerging PandemicThreats PREDICT project. We gratefully acknowledge the local support fromthe Thai Red Cross Society, Chulalongkorn University, Kasetsart University andthe Department of National Parks Wildlife and Plant Conservation. The views

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expressed in this manuscript are those of the authors and do not reflect theofficial policy or opinions of the US Department of Defense or Departmentof the Navy.

Author details1World Health Organization Collaborating Centre for Research and Trainingon Viral Zoonoses, King Chulalongkorn Memorial Hospital, Faculty ofMedicine, Chulalongkorn University, Bangkok, Thailand. 2Faculty of Forestry,Kasetsart University, Bangkok, Thailand. 3Department of National Parks,Wildlife and Plant Conservation, Bangkok, Thailand. 4Inter-DepartmentProgram of Biomedical Sciences, Faculty of Graduate School, ChulalongkornUniversity, Bangkok, Thailand. 5Royal Forest Department, Bangkok, Thailand.6EcoHealth Alliance, New York, USA. 7Naval Medical Research Center-Asia,Singapore, Singapore. 8Massachusetts General Hospital, Boston, MA, USA.

Received: 5 October 2014 Accepted: 25 March 2015

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